Method of separating particles from a filter

ABSTRACT

Particles that move at low Reynolds numbers are separated from a filter material on which they are adsorbed by subjecting the filter material, in a liquid medium, to agitation of sufficient magnitude to create turbulence and shear forces acting on the filter material such that the particles become desorbed from the filter material and become suspended in the liquid medium. The method may be used to recover biological particles, such as leucocytes and blood platelets, from filter materials on which they are retained by adsorption. The recovered particles may then be used in diagnostic testing and analytical techniques.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method of separating particles from a filtermaterial on which they are retained by adsorption and a method ofrecovering particles from fluids containing them. More particularly, theinvention relates to a method for rapidly and selectively recoveringbiological particles, such as leucocytes and platelets, in a form inwhich they can be used for many different purposes.

2. Discussion of Prior Art

There are many fluids in which leucocytes may occur: whole blood, bloodcomponents, milk, colostrum, urine, tissue and tumour disaggregates andexudates, lymph, ascites fluid, cerebrospinal fluid, bile, peritonealfluid, synovial fluid, seminal fluid, lacrimal fluid, interstitialfluid, hemolymph, saliva, tears, mantle fluid, bone marrow, coelomicfluid, glandular secretions, bronchoalveolar lavage, alveolar fluid,fluid from organs and tissues in culture, pathological discharges,discharges, mucus, pus and odematous fluid that accumulates in any spacewithin a living organism.

Much information can be obtained from measuring, for example, theproduction by leucocytes of free radicals and other reactive oxygencontaining species and of the enzymes produced and sometimes released byleucocytes when they become activated. These can be measured by adding asuitable luminogenic material, e.g., Pholasin (Registered Trade Mark),which will emit light in the presence of free radicals, certainoxidants, some reactive oxygen species and certain enzymes and any lightemitted would be a measure of the activity of the leucocytes. Many othertests, including non-luminogenic tests are also performed on leucocytes.However, it is often essential if such tests are to be performedaccurately, that the large majority of other cells, such as red bloodcells, which may be present in the fluid from which the leucocytes wereharvested are removed. It is also important in many cases to remove theplasma (or serum) and other fluid in which the leucocytes occur beforecarrying out many tests as components in the fluid may interfere withthe performance of many tests.

One approach to the problem of potential interference from plasmacomponents, such as complement, is to dilute a sample of whole blood atleast 1:500. However, this procedure reduces at the same time the numberof cells present in the sample by the dilution factor and, therefore,any measurable signal is also correspondingly reduced. As leucocytes, inwhole blood, are in relatively small numbers compared to the red bloodcells, any tests requiring large number of leucocytes isolated from redblood cells, or tests requiring the extraction of specific componentsfrom leucocytes, would still not be possible using samples of dilutedwhole blood.

There is a recurrent need selectively to separate biological particles,such as leucocytes or platelets, from blood, for example, for subsequentanalysis. There is also a need selectively to separate leucocytes fromother body fluids, for example seminal fluid, in order to obtain a puresample of sperm which are to be used in subsequent fertility tests.

There are known methods for separating biological particles, such asleucocytes, from blood, for example, for subsequent analysis or even fortreatment to insert a new gene, tag a radioactive label to the cell orother such procedure which involves removing leucocytes and thenreturning such modified cells to the whole organism or to an isolatedorgan or tissue. Such methods include multistep procedures involvingsedimentation with dextran, followed by separation on density gradients.These procedures involve centrifugation, mixing, incubating andsometimes lysis of unwanted red blood cells. They take a number of hoursto complete, involve skilled operatives and subject the cells touncontrollable variables which may inadvertently affect their subsequentresponse to analytical procedures (Boynum, A. (1968) "Isolation ofmononuclear cells and granulocytes from human blood". Scand. J. Clin.Lab. Invest. 21, Supple 97 (paper IV), 77-89). In addition the processhas to be conducted in a laboratory and thus cannot be used in generalmedical or veterinary practice, at the bedside, in the field or in anout-patient clinic for example.

In response to the need for a rapid, simple and reliable method thefollowing method was disclosed (Ferrante, A. and Thong, Y. H. (1980)"Optimal conditions for simultaneous purification of monocluclear andpolymorphonuclear leucocytes from human peripheral blood by theHypaque-Ficoll method". J. Immunol Methods, 35 109-117), which involvedlayering whole blood on to a mixture of Ficoll and sodium and/ormeglumine diatrizoate prepared to specific densities, centrifuging thetube and collecting layers of leucocytes which were washed withcentrifugation 2 to 3 more times. This `improved` method which, enabledleucocytes to be separated, washed and-ready for analysis in about 1 to2 hours still required a skilled operative and the need for acentrifuge. Also, it was not possible to carry out the proceduresimultaneously on more than a very few samples (usually not more than4). Furthermore, the method is not suitable for most bloods other thanhuman, and does not work efficiently on blood from people having certaindiseases, such as rheumatoid arthritis, juvenile rheumatoid arthritis ormicrocytic hypochromic anaemia. In addition, the method may fail or givevariable results if the patient was receiving aspirin, indomethacin,prednisone, aurothioglucose, or drugs for the treatment of bronchialcongestion, immunodeficiency anaemia or other diseases. An improvedmethod, designed to enable leucocytes to be separated from these`difficult` bloods was developed (Ferrante, A., James, E. W., Betts, W.J. and Cleland, L. G. (1982) "Rapid single step method for purificationof polymorphonuclear leuococytes from blood of patients with rheumatoidarthritis", Clin. exp. Immunol. 47, 749-752) in which the viscosity ofthe density medium was changed. The results of this improved method arestill variable and not useful for quantifiable and comparative work.

The mode by which leucocytes are separated using density gradients inthe prior art method is dependent upon the osmotic loss of water fromsedimenting erythrocytes into a slightly hyperosmotic gradient medium.As the erythrocytes sediment and lose water this leads to a dilution ofthe Hypaque-Ficoll and subsequent production of a continuous gradient oflower density which the granulocytes enter at the exclusion ofmononuclear cells and platelets. The production of the continuousgradient is dependent upon the erythocyte volume and usually requiresabout 5 ml blood to create a suitable gradient. A more recentimprovement has been disclosed by Kalmar, J. R., Arnold R. R.,Warbington, M. L. and Gardner, M. K. (1988) "Superior leukocyteseparation with a discontinuous one-step Ficoll-Hypaque gradient for theisolation of human neutrophils", J. Immunol. Meth. 110, 275-281, inwhich one density medium of specific gravity 1.114 and osmolality of458±10 mosM was used together with another medium of 1.077 specificgravity. The result was separation of leucocytes from 1 ml of wholeblood.

In all the prior art methods, even the "improved" ones, the cells aresubjected at times to adverse conditions. While it might betheoretically possible for a trained operative to work in precisely thesame manner at each separation, the differences between the blood ofvarious individuals especially during disease, make it impossible, insome instances, for the leucocytes to be separated from theerythrocytes. In addition, it is impracticable, even for trainedoperatives, to standardise the ways they perform the variousmanipulations involved in the lengthy procedure. The prior art methodsare therefore only suited to the separation of leuococytes from wholenormal blood and even then the sample volume required is not less than 1ml.

Knight Scientific Limited, in EP-A-0489602, disclose a method by whichleucocytes may be selectively removed from whole blood or any otherbiological fluid in which they occur by a rapid filtration techniqueusing a filter material having a critical wetting surface tensiongreater than 53 dynes/cm and capable of holding said leucocytes byadsorption. The filter material together with the adsorbed leucocytesmay then be subjected to a luminogenic material and introduced into aluminometer.

SUMMARY OF THE INVENTION

The present invention disclosed here bestows advantages over thatdescribed in EP-A-0489602. According to that document, the leucocytesremained adsorbed onto the filter and all subsequent analyses werecarried out in the presence of the filter. In order to carry outreplicate analyses, separate filtration devices are required and thisincreases the cost of the tests. Other determinations, such as tissuetyping, histological analysis and other biochemical tests or thesubsequent extraction from the leucocytes of enzymes and othersubstances including DNA and RNA is impeded by the presence of thefilter.

The particles that may be adsorbed onto a filter in the prior artfiltration technique are very small compared to the filter. In general,such particles have a diameter of up to 100 μm. Leucocytes are muchsmaller and have diameters of about 15 μm. Such small particles (i.e.,having a diameter less than 100 μm) are known to behave very differentlycompared to larger objects, such as the filters which have a diameter ofat least a few millimetres, when placed in a turbulent fluid. Thepresent invention is based on our discovery that this difference can beexploited in a method according to which such particles may be separatedfrom filters on which they are adsorbed.

The basis for the difference in behaviour of small particles compared tolarge objects in a liquid medium under flow may be explained withreference to the fact that the particles are of dimensions at which lowReynolds numbers dominate. Reynolds number is defined as:

    Re=ρUL/μ

where ρ is the density of the fluid, U is the velocity of flow past theobject (or object through the fluid), L is some characteristic length ofthe object (usually the longest dimension parallel to the flow) and μ isthe dynamic viscosity of the fluid. The Reynolds number is a purenumber, without units, and its significance is relevant to the mechanismby which the adsorbed particles are liberated from the filter.

The Reynolds number approximates to the forces due to inertial effectsrelative to the forces that arise from the effects of viscosity.Knowledge of the Reynolds number thus allows one roughly to establishthe kinds of forces which will dominate during flow.

As a rough generalization, the Reynolds number of an object is about 100times the length or diameter of the object (in cm) times the speed offlow (cm/sec). At low Reynolds number 40 or less but generally 10 orless, inertial forces are small relative to viscous forces.

For objects of a given size and shape the Reynolds number is the samefor a given fluid. However, if the dimensions of the objects aresufficiently different so that one object(s) has low Reynolds number(s)compared to the other, then the effect of turbulent flow on both objectswill be different.

This situation is what prevails in the method herein described ofliberating the adsorbed particles from the filter. The adsorbedparticles are generally less than 100 μm in diameter, (the leucocytesare about 15 μm) and, therefore, move at low Reynolds number whereas thefilter, is at least a few millimetres in diameter. At these dimensions,the effect of agitating the fluid so as to create turbulence and shearforces (in many directions) on the filter results in the filter movingrelative to the particles. The particles are left in suspension as thefilter, subjected to inertial forces, moves. Although the best resultsare obtained when turbulent flow prevails, the particles, unlike thefilter, never experience turbulence and move smoothly in suspension inthe liquid.

One object of the present invention is to provide a means of removingparticles of low Reynolds numbers from a filter material to which theyare retained by adsorption.

Another object of the present invention is to achieve the removal ofadherent biological particles, such as leucocytes, from the filter insuch a way that they are not damaged in the process and are notactivated sooner than is required. A further object is to enable therapid recovery of a sufficiently large proportion of biologicalparticles in a medium in which other tests, especially those involvingthe addition of a luminogenic material, such as Pholasin (RegisteredTrade Mark), and the measurement of light emission in a luminometer, canbe carried out.

The present invention provides a method of separating particles thatmove at low Reynolds number(s) from a filter material on which saidparticles are retained by adsorption which comprises locating the filtermaterial having the adsorbed particles thereon in a liquid medium in avessel, subjecting the filter material and the liquid to agitation ofsufficient magnitude to create turbulence in the vessel thereby settingthe liquid and filter material into turbulent motion and creating shearforces on the filter whereby the particles become suspended in theliquid and then separating the liquid containing the suspended particlesfrom the filter material.

According to the present invention, there is also provided a method ofrecovering biological particles from a fluid in which the particles arepresent which comprises passing the fluid containing the particlesthrough a filter material capable of selectively retaining the particlesby adsorption whereby the particles are adsorbed onto and retained bythe filter material, and separating the particles from the filtermaterial according to the above method.

The method of the invention can be used to separate particles, of a sizesmall enough to result in their moving at low Reynolds number, fromfilters onto which the said particles are adsorbed. The method can, forinstance, be applied to the cleaning of filters to remove smallparticles, i.e., bacterial filters. Such a method may be combined withsonication, if required, and possibly coupled with intermittent backflushing of a small volume of liquid. However, the invention isparticularly suitable for removing biological particles, such asleucocytes, from filters on which they are retained by adsorption andthe invention will be further described with reference to the removal ofbiological particles.

By carrying out the method of the invention biological particles may berecovered in an undamaged state, i.e., in the same biochemical state aswhen they were first removed from the patient's body, in large numbersin an aqueous medium. The aqueous medium, containing the recoveredparticles, obtained by the present invention can be divided, ifnecessary, between separate tests and analyses thus precluding the needfor further, separate filtrations. Furthermore, the method makes itpossible to obtain a useful concentration of biological particles fortesting from only a small volume of sample fluid, i.e., less than 1 ml.This is of particular importance when larger volumes of sample fluid arenot available or cannot be taken from a patient's body withoutincreasing the risk to the patient's health to an unacceptable level.

The method is useful for separating all biological particles, e.g.,cellular matter, from a filter material to which the particles are boundby adsorption. The method has particular application in the separationof blood platelets and leucocytes since many testing and analyticalprocedures are carried out on these. Fluids, in which leucocytes mayoccur and from which they may be recovered, are listed above. Accordingto a preferred embodiment, the invention provides a rapid means ofrecovering leucocytes from whole blood, human milk or semen thusenabling more rapid analysis and diagnosis for the patient.

The filter material in the method of the present invention will be onecapable of selectively retaining the biological particles by adsorptionwhen a fluid containing the said particles is passed or drawn throughit. It may be comprised of one or more membranes of a suitable materialor may have a fibrous or particulate composition. Preferably, the filtermaterial will have a critical wetting surface tension greater than 53dynes/cm. Filter materials useful in the present invention are known inthe art.

There are a variety of devices that can be used to perform the simpleseparation of leucocytes from whole blood and the subsequent removal ofthe leucocytes from devices. They all embody a step in which the wholeblood (or another fluid) is allowed to fall upon a filter one type ofwhich is described in EP-A-0489602 and which has the ability selectivelyto retain leucocytes or platelets and allow the passage of erythrocytesor other non-adsorbed particles through the filter. The device might,for example, selectively remove leucocytes from milk or semen allowingnon-adsorbed epithelial cells or sperm to pass through the filter.

Prior to passing the fluid containing the biological particles throughthe filter material, the filter material is preferably wetted to preparethe filter material for the filtration step, although satisfactoryresults can be obtained even if the filter is not washed. Typically, aphysiological buffer is used to wet the filter material. This buffer maybe heated to an elevated temperature prior to use, i.e., up to 40° C.,although typically it will be at a temperature of about 37° C. Theincreased temperature will reduce the viscosity of the liquid, which hastwo effects. One is directly on the leucocytes, as they are moreretentive at higher temperatures. The other is to increase theefficiency by which the red blood cells, also small enough to move atlow Reynolds number, pass through the special filter. Filtration can bedifficult at low Reynolds number with very high pressure drops acrossthe filter and the reduction of viscosity during the filtration stepincreases the rate and efficiency of filtration. Filtration at arelatively high temperature is essential if the liquid to be filteredhas a high fat content, such as raw milk or blood shortly after a meal.Preferably, the filter material is wetted with phosphate buffered saline(PBS), made up with: 0.8 g NaCl; 0.02 g KCl; 0.115 g Na₂ HPO₄ ·2H₂ O;0.02 g KH₂ PO₄ per 100 ml solution. It is important, in the case wherethe fluid to be filtered is blood, that the PBS used to wet the filterdoes not contain any calcium or magnesium salts since the presence ofcalcium and magnesium on the filter may lead to clotting of the bloodand may also accidentally lead to activation of the leucocytes. If theleucocytes become activated during the process of cell separation, thenit is not possible subsequently to measure the activity of theleucocytes and relate such activity to various disease states.

Following any prior wetting step, the fluid containing the biologicalparticles to be removed by adsorption on the filter material isdelivered on to the filter material. Various techniques may be employedto deliver the fluid on to the filter material. Examples include the useof a syringe, pipette, automatic syringe and sampler, or deliverydirectly from the vein via a filter mounted within the blood collectingtube or syringe, or delivery from a tube or any other vessel into whichthe fluid has been temporarily stored prior to transfer to the filter.

The blood or other fluid can contain any anticoagulant, with EDTA beingespecially well suited to preventing the cells from becoming activated.Separation can be carried out from blood that does not contain anyanticoagulant but for this procedure to be successful, the blood mustnot be allowed to stand for a time long enough for the clotting processto have developed sufficiently, significantly to impede the flow throughthe filter. Preferably, in the case where the fluid is blood this shouldbe warmed, typically to at least 37° C., prior to delivery of the fluidto the filter material. We have found no difficulty in filteringroutinely volumes of blood as small as 250 μl and have also obtainedexcellent results using only 50 μl.

The fluid may be drawn through the filter, for instance by way of avacuum or pushed through the filter by direct pressure. Various devicescan be made to hold one or a number of samples of fluid and thefiltration step can be carried out manually or automatically, evencontrolled by a computer. As the fluid passes through the filtermaterial, the biological particles of interest, such as leucocytes,become adsorbed on to the filter material and, thereby, become retainedthereon. The non-adsorbed components of the fluid pass through thefilter material and may be collected for eventual disposal in acontainer. The container may be an absorbent pad as described inEP-A-0489602 although, preferably, is a closable vessel to enable safedisposal without contamination or leakage of the contents.

Following the first filtration step, which is completed in a fewseconds, the filter material together with the adsorbed biologicalparticles is preferably washed with a physiological buffer, such as aphosphate buffered saline containing no calcium or magnesium ions but towhich albumin at a concentration of 0.1% may have been added to goodeffect. This washing buffer may be preheated to an elevated temperaturewhich will be pre-selected depending upon the differential adsorption ofthe types of biological particles, e.g., leucocytes, during the step inwhich they are removed from the filter. In a typical embodiment, thewashing buffer will be preheated to a temperature of about 37° C. Thevolume of washing buffer used in the washing step will, in general, befrom 1 to 6 times, preferably about four times, the volume of the fluidsubjected to filtration. Essential to the procedure to ensure quiescenceof leucocytes and to prevent clotting is the absence of calcium andmagnesium salts from the washing buffer.

The filter material having biological particles adsorbed thereon is,according to the present invention, placed in a liquid medium which istypically aqueous and preferably sterile and is subjected to agitationof sufficient magnitude to create turbulence in the tube, or othervessel, setting the liquid and filter into turbulent or random motionand thus creating shear forces, in many directions, on the filter whilstleaving the particles, not affected by the turbulent flow, in suspensionin the liquid and then separating the liquid with liberated particles,from the filter. The liquid, preferably aqueous, will usually becontained in a receptacle and the filter material having adsorbedparticles will be transferred from the filter apparatus to thereceptacle and, thus, brought into contact with the liquid medium inwhich particle removal from the filter material will be effected. Theaqueous medium is, typically, a physiological buffer such as phosphatebuffered saline. The temperature of this medium when it contacts thefilter material may generally be from 0° C. to about 40° C. However, wehave found that the removal of the biological particles from the filtermaterial is facilitated by locating the filter material in an aqueousmedium having a low temperature. Preferably, then, the temperature ofthe aqueous medium, when it contacts the filter material and adsorbedparticles, will be less than 10° C., preferably from 1° C. to 8° C.,especially about 4° C. Reducing the temperature has the effect ofincreasing the viscosity of the liquid medium and thus reducing theReynolds number further.

The presence, in the aqueous medium, of a substance which countersadhesion between the particles, i.e., reduces the capacity of thebiological particles to adhere together, has been found to improve theremoval of the particles from the filter material. Examples of suchanti-adhesion agents include, but are not limited to, heparin, gelatinand albumin. A satisfactory concentration of heparin is 5 units ml⁻¹.Gelatin of low bloom (about 75 bloom) gives good results. The amounts ofanti-adhesion agents, gelatin and albumin present in the aqueous mediumis typically from 0.01 to 0.7% by weight, although good results havebeen achieved using about 0.1% by weight of the agent. Theseanti-adhesion agents may also increase the viscosity of the liquidmedium and, thus, reduce the Reynolds number of the particles.

If the adsorption of the particles on the filter is in part, or totally,dependent upon electrostatic charges or other interactions, i.e.,hydrophobic interactions, then removal of the particles in the processdescribed above may be enhanced by the inclusion of substances, such aszwitterions, to neutralise charge effects or in the case of hydrophobicinteractions, reduce the salt content of the buffer to reduce theaffinity. In some instances, the particles may have a coating whichtakes on a positive or negative charge at different values of pH. If thefilter material is coated with a substance that results in a charge thenthe particles may be made to adhere to the filter by filtering in thepresence of a buffer which creates charge on the particles opposite tothat on the filter. Changing the pH, of the removing buffer to a valueof pH that either results in no charge on the particle or reverses thecharge so that the particles now have the same charge as the filter, mayimprove the efficiency of removal of the particles on agitation.

The volume of the aqueous medium contacted with the filter medium andadsorbed particles in the receptacle will, in general, be from 1 to 10times the volume of the fluid filtered in the first instance.

To effect removal, or de-adsorption, of the biological particles fromthe filter material the filter material with the adsorbed particles,which is located in the aqueous medium, is subjected to agitation ofsufficient magnitude to create turbulence and shear forces (in manydirections) on the filter resulting in the filter moving relative to theparticles. The particles are left in suspension as the filter, subjectedto inertial forces, moves relative to the particles. Various means ofagitating the filter to attain sufficient turbulence to produce shearingforces in many directions on the filter are known. One such means is avortex mixer. For example, the filter may be placed into 500 μl removingbuffer contained in a small tube that can be conical, rounded or evenflat at the bottom. A commercially available vortex mixer involves arubber cup into which a tube is placed. The cup revolves in an eccentricorbit of small radius. A tube containing a liquid is pressed into thiscup; the tube is not allowed to spin but the contents within the tubespin and a vortex is created. However, creation of a vortex of liquid isnot essential for the successful removal of cells adsorbed on thefilter; if sufficient agitation is created so that the filter moves in aturbulent manner then the cells will also be removed. As the filterspins or moves in a turbulent manner, the cells are liberated during theagitation. The cells that were adsorbed onto the filter are less than100 μm in diameter (with leucocytes having an average diameter of 15μm). For such small particles, inertia plays an insignificant role intheir motion which is directed by forces acting upon them at aparticular moment and not by anything in the immediate past. Suchparticles are said to move at low Reynolds number (a dimension-lessnumber which is the ratio of inertial to viscous forces acting upon theparticles). The filter, however is of a dimension in which inertia playsan important role in its motion. Therefore when the filter and theadsorbed leucocytes or biological particles are subjected to thiseccentric agitation the cells are left in suspension while the filterupon which they had been adsorbed is subjected to inertia and movesrelative to the particles. Another way would be to subject the tube andits content to a vibrational frequency of the order of 50 Hertz frommains electricity. Such a vibrational frequency is attained by anelectric razor. Similarly vibration of a similar frequency can beobtained with a make and break circuit device from a DC supply. Thereare a number of other ways to create turbulence and shear forces on thefilter one of these is to rapidly oscillate the tube back and forth.

A yet further way of bringing about the separation of particles from afilter material on which they are retained by adsorption is bysubjecting the filter material, in a suitable vessel and in the presenceof aqueous liquid, to agitation created by the movement towards and awayfrom the filter material in the vessel of an agitation means, forexample a plunger or piston-like device. For instance, the filtermaterial and aqueous liquid may be located in a tubular vessel and theagitation means may take the form of a tube, e.g., formed of plasticsmaterial, having an external diameter which is smaller than the internaldiameter of the tubular vessel which plastics tube has a closed bottomend. By moving the agitation means, such as by manual operation, insidethe tubular vessel with alternating motion towards and away from thefilter material with particles adsorbed thereon, the filter material issubjected to turbulence and to shear forces which cause the particles tobecome desorbed from the filter material and become suspended in theaqueous liquid in the tubular vessel. In one embodiment, the closedbottom end of the plastics tube is a flat perforated plate or is formedfrom a woven or non-woven fabric which is pervious to the aqueous liquidbut which does not adsorb the particles so that the aqueous liquid maypass through the end of the plastics tube as this is moved into and outof the tubular vessel. According to another embodiment, the agitationmeans comprises a flat member which is attached to a rod. The flatmember may be formed of a perforated plastics plate or a plate ofanother suitable perforated material or comprises a flat frame retainingor partially covered by one or more pieces of a mesh or woven ornon-woven fabric of appropriate pore size to allow the passagetherethrough of newly suspended particles in the aqueous liquid but notthe filter material on to which the particles are initially adsorbed.The mesh or woven or non-woven fabric which is pervious to the aqueousliquid and the separated particles will not have the ability to retainthe particles by adsorption. The rod may be removably attached to theflat member, e.g., by screw thread means, and may thus be removed fromthe member when the separation of the particles from the filter materialis complete. The member, thus, remains in the vessel resting on thefilter material so allowing the use of the liquid containing thesuspended particles without interference from the filter material.

According to the above-described procedures, biological particles arereleased into the aqueous medium which can be decanted from, orotherwise separated from, the filter material. The filter material maythen be located into another fresh supply of aqueous medium and theprocedure repeated so as to remove any further particles from the filtermaterial. These further particles may comprise the same as, or adifferent sub-set of particles compared to, the first particles releasedfrom the filter material. When the filter material is sufficientlydepleted of most or all of the adsorbed biological particles it may bediscarded. The different extracts containing the de-adsorbed particlesmay be pooled or retained separately.

A small volume (e.g., 10%, 100 μl in a total volume of 1 ml) ofconcentrated calcium and magnesium salts (0.1 g anhydrous CaCl₂ ; 0.1 gMgCl₂ ·6H₂ O per 100 ml solution) may be added to the particles removedfrom the filter to restore the ions needed by the cells to respondnormally to stimuli of the kind used in various cell activation studies.Alternatively, calcium and magnesium salts at normal physiologicalconcentrations can be included in the removing buffer although theabsence of the salts during all the separation procedures prevents thecells from becoming inadvertently activated.

The entire procedure can be completed in less than 1 minute and devicesmay be designed to carry out the separation simultaneously on a numberof different samples. The separated particles can now be used in any waydesired. An example of a method that works exceptionally well is thetransfer of 100 μl aliquots of separated cells into cuvettes containing250 μl of a solution of Pholasin® in physiological buffer plus anadditional 100 μl of either buffer or drug. The preparation is placed ina luminometer and an effector, a stimulating agent such as phorbolmyristate acetate, is injected into the cuvette and light measured asthe leucocytes become activated.

The separated leucocytes can even be tagged with, say, a radioactivelabel, and introduced into an organism or excised organ or tissue. Ifsuch a procedure is undertaken then the separation must be carried outunder sterile conditions and the buffers and device are to be suppliedsterile.

DESCRIPTION OF THE DRAWINGS

The invention will be further described with references to theaccompanying drawings of which:

FIGS. 1A-1C show the device in its initial configuration beforefiltration of a fluid has commenced;

FIG. 2 shows the same device after filtration;

FIGS. 3A-3C show the device disassembled after filtration to release thefilter material.

FIGS. 4A-4C show a preferred embodiment of an apparatus suitable forcarrying out the method of the invention.

FIGS. 5 and 6 show alternative agitation means that may be used in theapparatus illustrated in FIG. 4A.

DETAILED DISCUSSION OF PREFERRED EMBODIMENTS

In FIGS. 1A-1C the device comprises a tube 1 having a ledge 2 runningaround the inner surface of the tube on which a filter 3 rests.Alternatively, the tube may be fitted with a perforated plate on whichsits a removable filter 3. The filter has a diameter equal to thediameter of the plate. A funnel-shaped receptacle 4, which may be closedby an openable cap 5, is attached to the tube for instance by means of ascrew thread provided at the bottom of the receptacle which engages witha complimentary thread provided at the top of the tube or which may be apush fit within tube 1. Other forms of attaching the receptacle to thetube are also possible, as is known in the art. The size of the filteris such that it is held in position by the receptacle when attached tothe tube.

More than one filter can be employed, for instance to collect differenttypes of biological particles on different filters, as is known in theart. The filter or filters are typically circular although other shapes,e.g., elliptical, square or rectangular may be used. The tube opens outinto a barrel 6 which is provided with a plunger 7 slidably disposedtherein. The plunger has a head 8 which makes an air-tight andfluid-tight seal with the internal sides of the barrel and a handle 9which extends through the end of the barrel 10.

In use, fluid containing the biological particles to be recovered, e.g.,blood, is added to the receptacle 4 and is drawn through the filter 3using the suction caused by withdrawing the plunger 7 in the barrel 6.While the biological particles are adsorbed on and, thus, retained bythe filter, the non-adsorbed portion of the fluid including particles ofsimilar size to those retained, such as the red blood cells, iscollected in the barrel 6. Following the filtration, a quantity of awashing buffer is added to the receptacle 4 and this is also drawnthrough the filter by further withdrawal of the plunger from the barrel.

After the washing stage has been completed, the device will have theconfiguration shown in FIG. 2 wherein the washing buffer and thenon-adsorbed portion of the fluid 11 is retained in the barrel.

Then, the receptacle 4 is disengaged from the tube 1, as shown in FIG.3A. The filter 3 now with adsorbed biological particles thereon is thusreleased and may then be placed in contact with an aqueous medium in areceptacle for the recovery of the particles (not shown). The barrelportion of the device containing the non-adsorbed fluid and washings maythen be disposed of safely without spillage of any of the contents.

The tube end of the barrel may be closed with a cap or lid (not shown)to ensure the security of the contents in the barrel. By disposing ofthe contents of the barrel in this way, the release of any pathogens orinfectious agents that may be present in the fluid is prevented.

By an appropriate modification of the device shown in FIG. 1, it will beseen that it is possible to deliver blood direct from a patient's veinto the filter for instance by adapting the receptacle to receive ahypodermic needle. The blood may, in such a case, be delivered to thefilter by venous pressure or by withdrawing it from the vein using theplunger in the barrel.

This new invention also embodies the safe collection of waste bloodproducts which can be carried out in a number of different ways. In themanual method, handling of blood or other fluids containing leucocytesis kept to an absolute minimum whereas with the automated version theoperator is spared any contact with the whole blood or its washings asthe entire procedure is carried out automatically.

The device can also be adapted to use the collected filtrate, forexample in the ease of sperm separated from contaminating leucocyteswhen the sperm can be used in other tests, in particular those designedto test the sperm cells in fertility tests. The interpretation of suchtests is frequently hindered by contaminating leucocytes in thepreparation, and this device can be used to produce a pure preparationof such particles.

One preferred embodiment of an apparatus which is suitable forperforming the method of the invention is illustrated schematically inFIG. 4. According to this, the apparatus is formed of a vessel 12 whichcomprises a first tube 13 and an agitation means 14 comprising a secondtube 15 which has an external diameter smaller than the internaldiameter of the first tube. The second tube 15 of smaller diameter isfitted with an O ring 16 to facilitate ease of motion of the smallertube within the larger tube while preventing liquid from leaking fromthe first tube during operation. According to an alternative embodiment(not illustrated) the second tube may just touch the inner walls of thefirst tube and the material from which it is made permits easy movementof the second tube within the first tube. Both the first tube and thesecond tube are typically formed of plastics material. The second tubehas a bottom end 17 which is perforated. Alternatively, the end of thesecond tube may be closed by a piece of mesh or woven or non-wovenfabric pervious to the liquid, such as a filter material. In such cases,care should be taken, however, to choose a fabric that has no adsorptiveproperties related to the particles to be separated according to themethod of the present invention. To operate the apparatus shown in FIG.4 to carry out the method, an appropriate quantity of liquid medium 18and a filter material 19 having particles retained thereon by adsorptionare added to the vessel. It is possible to use more than one filtermaterial having adsorbed particles so as to produce a more concentratedfinal suspension. The agitation means is then inserted into the vesseland is moved alternatively towards and away from the bottom of thevessel (as shown by the direction of the arrows in FIGS. 4A and 4B) sothat, as it moves towards the bottom of the vessel, the liquid passesthrough the perforations in the bottom of the second tube and thus,enters the second tube. The reciprocating motion of the agitation meanscreates turbulence in the liquid and shear forces which act on thefilter material so as to separate the particles 20 therefrom. Thethus-liberated particles 20 become suspended in the liquid in the vesselwhich liquid, as described above, is drawn through the pervious end ofthe second tube. When the reciprocating motion of the agitation meanshas been carried out for a suitable period of time such that separationof the particles from the filter material is complete the agitationmeans may be used as shown in FIG. 4C to retain the depleted filtermaterial at the bottom of the vessel to facilitate the removal and useof the liquid medium containing the suspended particles.

According to the preferred embodiment shown in FIG. 4C, both tubes,i.e., first tube 13 and the pushed down second tube 15, are sealed witha removable cap 21 through which is inserted tubing 22 to enable thedispensing of the suspension of particles.

Instead of using the agitation means as shown in FIG. 4, use may be madeof the alternative agitation means illustrated in FIG. 5 or FIG. 6. Theagitation means shown in FIG. 5 comprises a perforated plate 23typically formed of a plastics material. The plate is centrally attachedto a rod 24. The rod has an end which is screw threaded and which isadapted to engage with an internally threaded block 25 attached to theplate. After separation of the particles, the rod may be unscrewed toleave the plate in the vessel to retain the filter material. Instead ofa perforated plate, a mesh or fabric-covered frame 26, as shown in FIG.6, can be used in the agitation means.

EXAMPLE

A suitable filter was positioned in a device of the type illustrated inFIG. 1 and described above.

250 μl of warm (37° C.) washing buffer was drawn through the filter towet and warm the filter. The whole device can also be kept warm. Then250 μl warm blood (37° C.) was allowed in impinge onto the filter andthe blood was drawn through the filter as described previously. 1 ml ofwarm washing buffer was then drawn through the filter.

The filter was then removed from the support and placed in a smallcentrifuge tube (maximum capacity 1.5 ml) containing 500 μl coldremoving buffer. The tube was subjected to four×5 second bursts ofeccentric agitation at fixed speed 2 on a vortex mixer (Autovortex mixerSA2 Stuart Scientific).

The liquid containing the liberated leucocytes were removed from thetube using a pipette. Then, 500 μl of removing buffer were added to thetube containing the filter and the tube was subjected to two×5 secondbursts of eccentric agitation at fixed speed 2 on the vortex mixer. Theliquid was removed from the tube by pipette and combined with the liquidrecovered from the first treatment. The total volume of liquid recoveredwas between 900-950 μl. From a leucocyte count it was determined that upto about 90% of the particles retained from the whole blood during thefiltration process could be recovered.

We claim:
 1. A method of separating particles that move at a Reynoldsnumber no greater than 40 from a filter material on which said particlesare retained by adsorption which method comprises the steps of:locatingthe filter material having the adsorbed particles thereon in a liquidmedium in a vessel; subjecting the filter material and the liquid toagitation of sufficient magnitude to create turbulence in the vesselthereby setting the liquid and filter material into turbulent motion andcreating shear forces on the filter whereby the particles becomesuspended in the liquid; and separating the liquid containing thesuspended particles from the filter material.
 2. A method of separatingparticles that move at a Reynolds number no greater than 40 from afilter material on which said particles are retained by adsorption,which method comprises the steps of:locating the filter material havingthe adsorbed particles thereon in a liquid medium in a vessel;subjecting the filter material and the liquid to agitation of sufficientmagnitude to create turbulence in the vessel thereby setting the liquidand filter material into turbulent motion and creating shear forces onthe filter whereby the particles become suspended in the liquid; andseparating the liquid containing the suspended particles from the filtermaterial wherein the particles are one of leucocytes and bloodplatelets.
 3. A method of separating particles that move at a Reynoldsnumber no greater than 40 from a filter material on which said particlesare retained by adsorption, which method comprises the steps of:locatingthe filter material having the adsorbed particles thereon in a liquidmedium in a vessel; subjecting the filter material and the liquid toagitation of sufficient magnitude to create turbulence in the vesselthereby setting the liquid and filter material into turbulent motion andcreating shear forces on the filter whereby the particles becomesuspended in the liquid; and separating the liquid containing thesuspended particles from the filter material, wherein the liquid mediumis a phosphate buffered saline.
 4. A method according to claim 3,wherein the phosphate buffered saline also contains an anti-adhesionagent to reduce the capacity of the particles to adhere together.
 5. Amethod according to claim 4, wherein the anti-adhesion agent is selectedfrom gelatin, albumin and heparin.
 6. A method according to claim 3,wherein the phosphate buffered saline also contains one of calcium andmagnesium at a physical concentration.
 7. A method as claimed in claim1, wherein the filter material on which the particles are retained byadsorption and the liquid medium are subjected to agitation in thevessel by movement of an agitation means towards and away from thefilter material.
 8. A method according to claim 7, wherein the agitationmeans comprises a tube having a closed bottom end.
 9. A method ofseparating particles that move at a Reynolds number no greater than 40from a filter material on which said particles are retained byadsorption, which method comprises the steps of:locating the filtermaterial having the adsorbed particles thereon in a liquid medium in avessel; subjecting the filter material and the liquid to agitation ofsufficient magnitude to create turbulence in the vessel thereby settingthe liquid and filter material into turbulent motion and creating shearforces on the filter whereby the particles become suspended in theliquid; and separating the liquid containing the suspended particlesfrom the filter material wherein the filter material on which theparticles are retained by adsorption and the liquid medium are subjectedto agitation in the vessel by movement of an agitation means towards andaway from the filter material, said agitation means comprises a tubehaving a closed bottom end, wherein the closed bottom end of the tube isformed of one of a perforated plate, a woven fabric and a non-wovenfabric.
 10. A method of separating particles that move at a Reynoldsnumber no greater than 40 from a filter material on which said particlesare retained by adsorption, which method comprises the steps of:locatingthe filter material having the adsorbed particles thereon in a liquidmedium in a vessel; subjecting the filter material and the liquid toagitation of sufficient magnitude to create turbulence in the vesselthereby setting the liquid and filter material into turbulent motion andcreating shear forces on the filter whereby the particles becomesuspended in the liquid; and separating the liquid containing thesuspended particles from the filter material, wherein the filtermaterial on which the particles are retained by adsorption and theliquid medium are subjected to agitation in the vessel by movement of anagitation means towards and away from the filter material, saidagitation means comprises a flat member centrally attached to a rod. 11.A method according to claim 10, wherein the rod is removable from theflat member.
 12. A method according to claim 10 or 11, wherein the flatmember is a perforated plate.
 13. A method according to either claim 10or 11, wherein the flat member comprises a flat frame which is at leastpartially covered with a mesh or woven or non-woven fabric.
 14. A methodof recovering biological particles from a fluid in which the particlesare present which method comprises the steps of:passing the fluidcontaining the particles through a filter material capable ofselectively retaining the particles by adsorption whereby the particlesare adsorbed onto and retained by the filter material; and separatingthe biological particles that move at a Reynolds number not greater than40 from the filter material by:locating the filter material having theadsorbed particles thereon in a fluid in a vessel; subjecting the filtermaterial and the fluid to agitation of sufficient magnitude to createturbulence in the vessel thereby setting the liquid and filter materialinto turbulent motion and creating shear forces on the filter wherebythe particles become suspended in the fluid; and separating the fluidcontaining the suspended particles from the filter material.
 15. Amethod according to claim 14, wherein prior to passing the fluidcontaining the particles through the filter material the filter materialis wetted with a physiological buffer at 37° C. which contains nocalcium or magnesium ions.
 16. A method according to claim 14, whereinthe fluid containing the biological particles is blood at a temperatureof about 37° C.
 17. A method according to claim 14, wherein, followingthe filtration step, a washing buffer medium which contains no calciumor magnesium ions at about 37° C. is passed through the filter material.18. A method according to claim 14, wherein the filter material havingthe biological particles retained thereon by adsorption is transferredto a receptacle containing an aqueous medium at a temperature of lessthan 10° C.
 19. A method according to claim 18, wherein the temperatureof the aqueous medium is in the range of from 1° C. to 8° C.
 20. Amethod according to claim 19, wherein the aqueous medium has atemperature of about 4° C.
 21. A method according to claim 14, whereinthe filter material is located in a syringe having on one side of thefilter material a barrel provided with a plunger slidably disposedtherein and on the other side of the filter material a receptacle forthe fluid containing the biological particles and wherein the fluid isdrawn from the receptacle, through the filter material and into thebarrel by the withdrawal of the plunger.
 22. A method according to claim21, wherein the receptacle is connected to a tube inserted into a humanor non-human patient's body and the fluid is delivered, to the filtermaterial direct from the patient's body.
 23. A method according to claim22, wherein the fluid is blood and the receptacle is connected to a tubeinserted into the patient's vein.
 24. An apparatus for separatingparticles that move at a Reynolds number of no greater than 40, saidapparatus comprising:a vessel containing a filter material on which saidparticles are retained by adsorption; a liquid medium; and agitationmeans for agitating the contents of the vessel by movement towards andaway from one end of the vessel so as to create sufficient turbulenceand shear forces on the filter whereby said particles become suspendedin the liquid.
 25. An apparatus for separating particles that move at aReynolds number of no greater than 40, said apparatus comprising:avessel containing a filter material on which said particles are retainedby adsorption; a liquid medium; and agitation means for agitating thecontents of the vessel by movement towards and away from one end of thevessel so as to create sufficient turbulence and shear forces on thefilter whereby said particles become suspended in the liquid, whereinthe vessel comprises a first tube and wherein the agitation meanscomprises a second tube having an external diameter smaller than aninternal diameter of the first tube and wherein the second tube has aclosed end.
 26. An apparatus according to claim 25, wherein the closedend of the second tube which forms the agitation means is pervious tothe liquid medium.
 27. An apparatus for separating particles that moveat a Reynolds number of no greater than 40, said apparatus comprising:avessel containing a filter material on which said particles are retainedby adsorption; a liquid medium; and agitation means for agitating thecontents of the vessel by movement towards and away from one end of thevessel so as to create sufficient turbulence and shear forces on thefilter whereby said particles become suspended in the liquid, whereinthe agitation means comprises a flat member centrally attached to a rod.28. An apparatus according to claim 27, wherein the rod is removablefrom the flat member.
 29. An apparatus according to claim 27, whereinthe flat member is a perforated plate or comprises a flat frameretaining or at least partially covered by one or more pieces of a meshor a woven or non-woven fabric pervious to the liquid medium.
 30. Anapparatus for separating particles that move at a Reynolds number of nogreater than 40, said apparatus comprising:a vessel containing a filtermaterial on which said particles are retained by adsorption; a liquidmedium; and at least one agitator moving towards and away from one endof the vessel thereby agitating the contents of the vessel so as tocreate sufficient turbulence and shear forces on the filter whereby saidparticles become suspended in the liquid.